pGL3-BMP2荧光素酶报告基因载体的构建及鉴定

Long fragment of pGL3 cloning and it's eukaryotic expression vector construction

目的扩增BMP2基因下游110kb处的预测有增强子功能的非编码序列,将其插入pGL3-promoter载体,构建该长片段的真核表达系统。方法在所设计的上下游引物的5’端加入BamHⅠ酶切位点,采用聚合酶链反应(PCR)的方法从短指畸形患者外周血提取的DNA中扩增BMP2基因下游区域预测有增强子功能的一段长约5kb的非编码序列。首先将扩增的目标片段与PGEM-T载体连接,连接产物转化DH5α感受态细菌,经蓝白斑筛选出的阳性克隆通过电泳、酶切鉴定后进行测序验证是否为插入的目标序列,测序正确后提取重组质粒。利用限制性内切酶BamHⅠ同时酶切插入目标序列的质粒和pGL3-promoter载体,将酶切完全的目标序列与线性pGL3-promoter空质粒载体在Solution I快速连接酶的作用下进行连接反应,构建目标序列的真核表达载体。结果经过酶切和测序证实含有4.8kb长的目标序列的分子克隆和真核表达载体构建成功。结论实验成功克隆了BMP2基因下游区域预测有增强子功能的一段长约5kb的非编码序列,并构建了pGL3-promoter-enhancer真核表达载体,为进一步验证该序列可能的调控功能奠定了基础。

Objective： According to previous reports, we amplified an approximately 4. 8kb non -coding sequence with predicted enhancer regulatory function located at 110kb downstream of BMP2 and cloned it into the pGL3 - promoter vector to construct the eukaryotic expression system of the whole length fragment. Methods： Adding the restriction sites of the BamH I to the 5＇site of the designed primers and received the target sequence by PCR performed from the DNA of the normal individuals. First, we cloned the target sequence into the PGEM - T vector and screened the positive cloning by IPTG - Xgal trial. Extracted the plasmid and identified whether inserted the target sequence by enzyme reaction and electropboresis. Then using BamH I to digest the positive plasmid and pGL3 - promoter vector respectively to gain adhesive end and performed the coupled reaction of each other by rapid ligase Solution I . Finally the pGL3 - promoter eukaryotic expression vector was constructed. Results ： From the BamH I digesting and sequencing identification we successfully cloned the 4. 8kb length fragment into the pGL3 - promoter vector and constructed the eukaryotic expression vector. Conclusions ： Our experiment performed two molecular cloning process and had a success in constructing for pGL3 - promoter - enhancer expression vector contained a 4. 8kb non - coding sequence with predicted cis - regulatory function located at 110kb downstream of BMP2, and established the further study foundation of the probable function.